Purpose :
Supplementing human meibomian gland epithelial cells (HMGECs) with omega-3 (n-3) and -6 (n-6) fatty acids has been reported to increase nonpolar lipid production, but the n-6:n-3 ratio may be more relevant physiologically. Higher ratios may exacerbate inflammatory disease, and lower ratios may mitigate disease. Therefore, an investigation evaluating n-6:n-3 and their effects on the lipidomic output of HMGECs in the presence of a known disease-inducing compound, 13-cis retinoic acid (RA), was performed.

Methods :
HMGECs were cultured with and without 0.5 µM 13-cis RA and 100 µM of omega fatty acids in the following ratios: LOW (4:1), MED (8:1), or HIGH (16:1). Docosahexaenoic acid (n-3), eicosapentaenoic acid (n-3), and arachidonic acid (n-6) were used. After two days, lipids were extracted by a modified Folch technique and directly infused into a Triple TOF 5600 mass spectrometer (Sciex, Framingham, MA) with electrospray ionization. MS spectra were acquired in both positive and negative modes. Lipid species were identified by characteristic m/z ratios.

Results :
Across all conditions, there were 39 identifiable peaks that are known to be meibum-relevant lipids (MRLs). Triacylglycerols (TAGs, nC = 48-56), wax esters (WEs, nc = 34-44), and cholesteryl esters (CEs, nc = 18-26) were the most abundant. In the absence of 13-cis RA, there were no differences in any MRLs between the LOW, MED, and HIGH conditions. With 13-cis RA, there was a significant increase in ten TAGs (mean fold change = 2.36, p < 0.027 for all) and one CE (fold change = 2.49, p = 0.024) relative to controls. Concurrent supplementation with both 13-cis RA and LOW, MED, or HIGH yielded 23 common MRLs. HIGH yielded the greatest expression for 22 of these 23 with significance attained for one WE, one CE, and five TAGs (p < 0.05 for all). Cells treated with HIGH and 13-cis RA showed a further increase in three TAGs (p < 0.029 for all) relative to 13-cis RA alone.

Conclusions :
Variations in n-6:n-3 in isolation do not independently induce meibum-relevant lipidomic changes from HMGECs. Treatment with 13-cis RA primarily results in triacylglycerol upregulation, which may be a meaningful biomarker of meibomian gland disease. Further, high n-6:n-3 seems to potentiate this triacylglycerol upregulation, while low n-6:n-3 seems to have minimal modulating effect.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.